Decellularized Wharton’s Jelly from human umbilical cord as a novel 3D scaffolding material for tissue engineering applications

<div><p>In tissue engineering, an ideal scaffold attracts and supports cells thus providing them with the necessary mechanical support and architecture as they reconstruct new tissue <i>in vitro</i> and <i>in vivo</i>. This manuscript details a novel matrix derived from decellularized Wharton’s jelly (WJ) obtained from human umbilical cord for use as a scaffold for tissue engineering application. This decellularized Wharton’s jelly matrix (DWJM) contained 0.66 ± 0.12 μg/mg sulfated glycosaminoglycans (GAGs), and was abundant in hyaluronic acid, and completely devoid of cells. Mass spectroscopy revealed the presence of collagen types II, VI and XII, fibronectin-I, and lumican I. When seeded onto DWJM, WJ mesenchymal stem cells (WJMSCs), successfully attached to, and penetrated the porous matrix resulting in a slower rate of cell proliferation. Gene expression analysis of WJ and bone marrow (BM) MSCs cultured on DWJM demonstrated decreased expression of proliferation genes with no clear pattern of differentiation. When this matrix was implanted into a murine calvarial defect model with, green fluorescent protein (GFP) labeled osteocytes, the osteocytes were observed to migrate into the matrix as early as 24 hours. They were also identified in the matrix up to 14 days after transplantation. Together with these findings, we conclude that DWJM can be used as a 3D porous, bioactive and biocompatible scaffold for tissue engineering and regenerative medicine applications.</p></div

Transplantation and culturing of WJMSCs on DWJM.

<p><b>A</b>) Confocal microscopy images of DWJM and WJMSCs on DWJM after 2 hours (upper panel), 1 day (center panel), and 2 days (lower panel) post- cell seeding. The cells are labeled with calcein acetylmethyl (AM) that stains the live cells in green. Dual beam imaging of <b>B)</b> DWJM and <b>C)</b> DWJM seeded with WJMSCs for 1 week. The Everhart-Thornley detector (ETD) is a standard secondary electron detector used in scanning electron microscopy to study topography, while the circular backscatter (CBS) is a backscatter detector that reveals lipid content when samples are stained with osmium tetroxide (OT) (red/orange). Images have been pseudo-colored to enhance definition proportional to secondary electron signal for ETD. (Scale bar is 20 μm.) DWJM appears to be a fibrous interpenetrating network with varying pore sizes, while WJMSCs were arranged along the fibers of DWJM.</p

WJMSCs transplantation into an <i>in vivo</i> animal model.

<p>A) Mice with cranial defect, B) mice with cranial defect and DWJM, C) mice with cranial defect and DWJM 14 days post-surgery. Arrows in A represent the defect, B shows the DWJM and C is the defect and DWJM 14 days post-surgery. D) IVIS imaging of the mice post—surgery—1) Mice with DWJM 24 hours post- surgery; 2–6) designates mice 14 days after the surgeries. D2 is mice without any intervention, D3 and D4 are mice with the defect alone, and D5—D6 represent mice with defect and DWJM. The red circles indicate the defect sites and the inset images are a higher magnification of the defect site in mice. <i>The green fluorescence signal at the defect site signifies the migration of the GFP positive cells into the defect</i>. Images E-J represent the histology images of bone specimen with DWJM 14 days post-surgery, with image E) hematoxylin-eosin stained (H&E) section of DWJM tissue specimen 24 hours post-surgery, and image F depicts GFP immunohistochemistry staining of the same. Images G-J represent DWJM sample 14 days post-surgery with G, H and I being H&E stained sections of DWJM viewed at different magnifications as indicated in the figure. J represents the GFP immunohistochemistry of the section in image I. The arrows in image F, J represent GFP positive cells.</p

Relative fold change in the mRNA levels of the indicated genes.

<p>Panel A-F are WJMSCs on DWJM with A) Cell adhesion genes, B) Chondrogenic genes, C) Adipogenic genes, D) Myogenic genes E) Osteogenic genes, F) Apoptosis and proliferation genes. Panel G-L are BMMSCs cultured on DWJM with G) Cell adhesion genes, H) Chondrogenic genes, I) Adipogenic genes, J) Myogenic genes K) Osteogenic genes, L) Apoptosis and proliferation genes. Relative fold- change is represented on the y-axis and the genes were represented along the x-axis. The horizontal line represents the gene expression of cells before seeding at Day 0. (* Represents statistical significance p<i><0</i>.<i>05</i>.<i>)</i></p

MSC characterization by flow cytometry.

<p>A) Wharton’s jelly mesenchymal stem cell (WJMSCs) and, B) bone marrow mesenchymal stem cell (BMMSCs). All MSCs stained positive for CD90 by fluoroscein isocyanate (FITC), CD105 by phycoerythrin (PE) and CD73 by allophycocyanin (APC); and they were negative for hematopoietic markers CD45, CD34, CD14 or CD11b, and CD20 as analyzed by Cell Profiler (CP) software (Broad Institute).</p

Assessing WJMSC viability and proliferation when seeded on DWJM.

<p>A) Alamar blue assay to assess the viability of cells seeded on the matrix and B) Cell migration assay performed using trans-wells with cells alone (control) and cells migrating towards DWJM, (* Indicates statistical significance <i>p < 0</i>.<i>05)</i>.</p

Quantification of DWJM.

<p>A) DNA quantification study performed on the matrix before decellularization and after decellularization. DWJM showed significantly less DNA compared to the native WJ matrix before decellularization. B) Glycosaminoglycan content assessment of the matrix before and after decellularization. (* Indicates statistical significance (<i>p</i> < .05)).</p